Project Summary Head and Neck Squamous Cell Carcinoma (HNSCC) is a very prevalent cancer, affecting approximately 630,000 people per year. While every cancer has unique characteristics, HNSCC is especially heterogenous due to a variety of tissue origins and mutations. Inactivating mutations in Notch1 occur in 15-20% of HNSCC cases, a marked difference from the prevalence of Notch1 amplifications in other cancers. While Notch1 mutations are especially aggressive and associated with poor prognosis, we have also seen that they are associated with reduced expression of co-inhibitory receptors on immune cells. While co-inhibitory receptors are associated with immune exhaustion, they are also a natural consequence of immune cell activation. The reduced expression of inhibitory receptors in tumor-infiltrating lymphocytes is likely a sign of reduced immune activation. We will investigate the effect of Notch1 loss on the immunosuppressive environment in HNSCC and determine how this important signaling molecule might be impacting immunosurveillance. On the other side of the tumor-immune interaction, the other important question remains how to best block immune inhibition. Anti- PD-1 therapy has been a popular new treatment, but is only effective in 10-20% of cases. However, PD-1 is not the only inhibitory receptor on immune cells, and others such as lymphocyte activation gene-3 (LAG-3) are equally important. We will investigate how dual inhibition of these receptors can be optimized with respect to timing and strategic targeting of different receptors. The findings of these studies are expected to develop new approaches to cancer immunotherapy and optimizing strategies to existing immunotherapy treatments.